Disclosed are a holding tray, a substrate alignment system using the same and a method thereof. More specifically, the present invention relates to a holding tray for substrate capable of accomplishing high-precision alignment and conducting a stable deposition. A holding means is included in at least one side of the substrate to hold and support the substrate in a manner that the substrate is vertically held and supported on a flat surface of the holding tray during a vacuum process.The holding tray according to the present invention, the substrate alignment system using the same, and the method thereof include a substrate on which a deposition is made, a frame formed to receive the substrate, a tray formed to receive the frame, and at least one holding means formed to hold the substrate on the frame.The holding tray for substrate according to the present invention, the substrate alignment system using the same, and the method thereof are useful to accomplish high-precision alignment and conduct a stable deposition process due to the stable vertical arrangement of the substrate during the deposition process.